The use of a focused laser beam to create a sub-micron diameter self-healing pore in the plasma membrane of a cell (photoporation/optoporation), for the selective introduction of membrane impermeable substances (optical injection/optoinjection) including nucleic acids (optical transfection), is a powerful technique most commonly applied to treat single cells. The membrane of a healthy cell is impenetrable to large polar molecules. The ability to overcome this barrier and inject a foreign material, such as nucleic acids (e.g. DNA, mRNA, interference RNA), a stain or a drug for example, into a living biological cell without damaging the integrity of the cell or the agent, is of interest to a wide range of applications in biology and medicine.
A diverse range of methods exist for permeabilizing the membrane of a cell for the insertion of foreign material including: the insertion of micron-sized pipettes (microinjection); application of electric fields (electroporation); ballistic insertion of coated nanoparticles (gene gun); transportation of therapeutic agents encapsulated in lipid- (lipofection) or polymer-based particles; viral delivery; pore formation or permeabilization using acoustic waves (sonoporation); and using laser fields to open a transient pore in the membrane (photoporation). Of these, photoporation has successfully been demonstrated on a wide range of both animal and plant cell types and has numerous advantages. However, photoporation approaches to date have been limited to low throughput, small-scale studies, as they typically require manual sequential dosing of individual cells.
One system that starts to address the issue of throughput in photoporation is described by Marchington et al. in Biomedical Optics Express 1, 2, 527 (2010). This system has a microfluidic chip that is used to deliver cells through a focused femtosecond laser beam for photoporation, enabling cells to be targeted in an automated approach. The beam is focused to a diffraction limited spot using an external microscope objective, and cells pass through the focus in an orthogonal direction to the direction of laser propagation. Doing this allowed throughputs of one cell per second to be achieved. However, due to the requirement for the cells to be exposed to the beam for 1-10's of milliseconds, the throughput is still limited.